Fig. 2: Screening of pathway missing enzymes. | Nature Communications

Fig. 2: Screening of pathway missing enzymes.

From: Construction of a synthetic metabolic pathway for biosynthesis of 2,4-dihydroxybutyric acid from ethylene glycol

Fig. 2

Purified N-terminal his-tagged candidate NAD(P)-dependent D-threose dehydrogenase (a) and D-threonate dehydratase (b) enzymes were tested on natural and synthetic substrates. Absence of measureable activity is indicated by an asterisk (*). Results represent the mean of two biological replicate experiments. Individual data points are shown as coloured black dots. For analysis of candidate D-threose dehydrogenases, substrate concentrations of co-factor and sugar were 10 mM each. All candidate D-threonate dehydratase enzymes were tested on D-threonate and the respective natural substrate (Ec.IlvD, Ss.IlvD: 2R-dihydroxyvalerate; Xc.FucD, Pp.FucD: L-fuconate; Bj.TarD: D-tartrate; Aa.AraD, Hh.AraD: D-arabinonate; Ec.UxaA: D-altronate) using the semicarbazide assay which detects 2-keto acids. Substrate concentrations were 10 mM (except for Aa.AraD and Hh.AraD which were assayed on 1 mM of their natural substrate D-arabinonate). Enzymes with activity on D-threonate were further verified to produce OHB in a coupled assay with OHB reductase Ec.Mdh5Q (see Supplementary Fig. 4). Source data are provided as a Source Data file.

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